1. Field of the Invention
The present invention relates to purification, isolation, concentration and separation of organic or biological substances by a novel continuous flow isoelectric focusing method (contifocusing) and apparatus (contifocuser), a technique which employs an electrically low-conductive field for concentration or separation of charged substances. Centrifugation, filtration, absorption steps or carrier electrolyte or ampholyte buffers, to establish suitable pH gradients, are not used.
2. Description of the Related Art
Isoelectric focusing is a known technique for separating charged molecules such as proteins from a mixture containing them, using the principle that such charged amphoteric molecules have a zero net charge at a particular pH, the so-called isoelectric point (pI value). The compounds will migrate to the pH of their own isoelectric point if they are subjected to an electric field, and are separated accordingly.
Methods and apparatus for static isoelectric focusing are known, e.g. from WO 79/00942; EP-A-256552 and U.S. Pat. No. 5,256,269. A shortcoming of this type of arrangement is the small quantity to be purified, and its collection in a batch-wise operation. The technological background of the present invention is described in a continuous flow electrophoresis apparatus in U.S. Pat. No. 4,465,582 (1982) which employs a controlled fluid flow in the electrode chambers, which flow is running parallel but separate from the flow in the xe2x80x9cworkingxe2x80x9d chambers. U.S. Pat. No. 5,160,594 discloses an isoelectrofocusing apparatus having a forced recirculation in every cell, i.e. a recycling isoelectric focusing process. Recycling is not used in the present invention. The US patent employs a plurality of devices for preventing mixing of semi-purified and crude amphoteric substances. Additional cooling is required in every cell to reduce ohmic heating. Strong acids and bases, a catholyte or anolyte, are confined to the electrode chambers by ion-selective membranes to repel the negatively or positively charged ions and to establish suitable pH gradients. As opposed to U.S. Pat. No. 5,160,594, the present invention uses ion-selective membranes for different purposes, namely to selectively permit passing of low-molecular substances towards the electrode space for discarding from the electrode outflow port. All prior art devices require carrier ampholytes for the working chambers, which have a relatively low conductivity and a buffering capacity. U.S. Pat. No. 5,160,594 describes a maximum conductivity up to 600 xcexcS/cm, which is far less than the conductivity up to 5000 xcexcS/cm of the present invention. Like other prior art apparatus it is not designed for purification on an industrial scale and, in addition, U.S. Pat. No. 5,160,594 does not provide examples of substances to be purified.
A continuous flow isoelectric focusing method and apparatus, without using carrier ampholyte buffers but for use on an industrial scale, are disclosed in Hungarian patent 210,584 (1992). It describes an upward flow system through the chambers by gravitation into the separation chambers up to the level of the outflow port. The system includes two non-conductive solution chambers where the second is having two or more solution outflow ports and a passageway for a downwards flow of the solution from the first chamber into the bottom of the second chamber. A pair of electrodes are located in the second chamber. A plurality of adjacent partition members are located in the second chamber and extended there across in order to provide substantially convection-free solution and current flow pathways in the horizontal direction. A shortcoming of HU 210,584 is the fact that in the inflow solution chamber a gradient can be formed by itself. The inflow of the solution by dropping is difficult to control. The upward flow system of the Hungarian patent is less vulnerable to intrinsic gradient formation, alike in the present invention, but the two-chamber system is complicated and difficult to construct. In addition, separation of organic solutions occurs at both opposite sides of the second separating chamber close to the electrodes. Inorganic ions are easily mixing with the single fractions of inorganic or biological substances to be separated. A second separation step is frequently required.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
The present invention provides a contifocuser for separating substances in an electrically conductive solution, which apparatus is made from electrically non-conductive material and includes a brick-form separating chamber having one liquid inflow, two electrode space outflows and one or more working solution outflow ports and a passageway for upward flow of the solution from the bottom up to top outflows. The ratio of the height, the width and the depth of the separation chamber can be e.g. 3:2:0.2. A pair of electrodes are located in vertical position along both opposite most narrow walls of the separating chamber. They are adapted to be coupled to an external direct current (DC) power supply.
The apparatus is divided into three parts by two partitions made from ion-selective membranes. These ion-selective membrane partitions are situated in vertical position at both sides of the apparatus near the electrodes, so that they each separate about {fraction (1/25)} to {fraction (1/15)} parts from both ends. Both vertical ion-selective partitions have an opening at the bottom with a diameter of one half of the inflow pipe of the apparatus. In particular, the one enclosing the cathode space is a cation-selective membrane and the other is an anion-selective membrane. The ion-selective membranes have a specificity chosen to permit the transfer of either cations or anions in order to be eliminated in the waste outflow ports. At both ends, the waste outflow openings are located near the electrodes at {fraction (1/40)} to {fraction (1/10)} from the top rim of the apparatus. The working outflows, for collecting the substances to be purified, are situated at the same level in the middle. Advantageously, the contifocuser comprises at least three of said outflow ports. Between the outflow ports, except in the electrodes spaces, are dividing vertical partitions made from electrically non-conductive material, closed in their upper quarter and with small openings from bottom up to e.g. three quarters of their height.
As a further improvement, a plurality of adjacent alternating zigzag-shaped or U-shaped or multiple U form partition members may be located in the remaining space of the apparatus between the above-mentioned partitions, but not in the electrode spaces. These are also made of electrically non-conductive material and are perforated or made from porous material permeable for ions and molecules in both directions, and extend from the bottom up to the lowest level of the working outflow port and across the chamber. The partition members are placed freely over the entire width of the apparatus, and are the novel means to provide a substantially convection-free solution but provide an undisturbed flow of electrolytes or amphoteric particles in the horizontal direction. The perforated membranes are in a spaced-apart relationship in order to provide an undisturbed vertical flow. This invention has important advantages over prior art devices in that it can process up to 3,600 L/day of earthworm enzymes (prior art according to HU 210,584: 280-300 L) producing 80,000-170,000 U. of purified enzyme per L/day (HU 210,584: 60,000-70,000 U.).
Another advantage of this invention is that the solution or suspension may have a conductivity in the range of 50-5000 xcexcS/cm, instead of 50-2000 xcexcS/cm of the prior art devices (U.S. Pat. No. 5,160,594 describes in the tables a conductivity up to 600 xcexcS/cm). In this invention, the solution flows through a chemically inert passageway of said apparatus, vertically upwards through a plurality of vertical flow paths formed between the two vertical ion-permeable membrane partitions which separate the spaced electrodes from the working space in-between. A small part of the solution flows horizontally through small openings in these partitions. A bias voltage is applied between the electrodes which results in a separation of the electrically charged components in horizontal direction across the apparatus towards the cathode or the anode. The components are collected as they exit through the outflow ports located near the upper part of the apparatus. The small and low M.W., and charged components, i.e. metals and ions, move to the electrode spaces. They exit the apparatus through waste outflow ports, and are therewith separated from the higher M.W. substances which move to the centre of the apparatus.
In this invention, the target substance comprises at least one zwitterionic species characterised by a pI value, wherein said species is surrounded by amphoteric impurities. A pH gradient is formed across the-apparatus by said impurities when the bias potential is applied. In the pH gradient, the zwitterionic species are driven to a point with a pH corresponding to the pI of said species, and is neutralised. Thereafter, the substance flows in the vertical direction to an outflow port in the upper part of the apparatus.
In order to increase the capacity, two contifocusers can be attached to each other in a twin configuration, and are separated by a waterproof vertical cross-wall (17). The xe2x80x9ctwin-focuserxe2x80x9d has common electrode spaces (2) at both ends of the apparatus, and one pair of electrodes serves for both contifocusing chambers. The twin-focuser has a common inflow port (7) in the centre-bottom of the apparatus where the incoming flow is equally divided between both contifocusers. From both electrode spaces one or two electrode outflows can be arranged (11). Twin-focuser has at both sides working outflow ports (12) as in the original contifocuser. The twin-focuser has a double flow rate and double separation capacity whilst the electrical energy requirement is increased by one-third only.
Purification, isolation, and concentration of biological substances on an industrial scale is described. Centrifugation, filtration, absorption steps or additional carrier ampholyte buffers to establish suitable pH gradients, are not necessary. Multiple dividing and perforated partition members, permeable for particles in both directions, are placed vertically over the entire width of the apparatus as the novel means to provide a substantially convection-free solution, and to create an undisturbed vertical fluid flow.
The contifocuser is designed for cost-effective purification of e.g., plasmids, peptides, amino acids, enzymes, immunoglobulins, antigens for diagnostic testkits which are manufactured by combining/mixing with further testkit components, or for preparing viral or bacterial (subunit) vaccines which are manufactured by combining/mixing with further vaccine components, allergens, and for isolating cell populations or milk-, blood-, or urine-borne recombinant products. The contifocuser can further be used for elimination of impurities or toxic substances from yeast or bacterial fermentation processes, antibiotics, organic solvents, drinking water and beverages.
The contifocuser can also be used for isolating or purifying products which are expressed by or extracted from a microorganism (including bacteria, fungi, yeasts, algae and other mono- or oligocellular organisms), animals (including mammals, insects, invertebrates) or plants (including seaweeds, plankton, mushrooms, mosses, herbs and higher plants), or parts or derivatives thereof. Such products include microbial expression products, substances produced by transgenic mammals such as cows and pigs producing medicinal products in their milk, and substances extracted from forestry and plantation products. Examples are natural colours, pesticides, lignin, polyols such as xylitol, carbohydrates, oligo- and polysaccharides such as xylans and microcrystalline cellulose, dietary and other fibres, glycoproteins, and lipoproteins.
Most of the biological substances separated or purified by the apparatus and method according to the present invention are amphoteric substances, although the apparatus is not limited to separating or purifying amphoteric substances and can include ions, heavy metals, etc.
The present invention also provides a hydrolytic enzyme or mixture of enzymes purified using the apparatus of the present invention and methods for using such a purified hydrolytic enzyme or enzyme mixture.